Differential reinforcement of cGAS-STING pathway-involved immunotherapy by biomineralized bacterial outer membrane-sensitized EBRT and RNT

Radiotherapy (RT), including external beam radiation therapy (EBRT) and radionuclide therapy (RNT), realizes physical killing of local tumors and activates systemic anti-tumor immunity. However, these effects need to be further strengthened and the difference between EBRT and RNT should be discovered. Herein, bacterial outer membrane (OM) was biomineralized with manganese oxide (MnO2) to obtain OM@MnO2-PEG nanoparticles for enhanced radio-immunotherapy via amplifying EBRT/RNT-induced immunogenic cell death (ICD) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) activation. OM@MnO2-PEG can react with H2O2 and then gradually produce O2, Mn2+ and OM fragments in the tumor microenvironment. The relieved tumor hypoxia improves the radio-sensitivity of tumor cells, resulting in enhanced ICD and DNA damage. Mn2+ together with the DNA fragments in the cytoplasm activate the cGAS-STING pathway, further exhibiting a positive role in various aspects of innate immunity and adaptive immunity. Besides, OM fragments promote tumor antigen presentation and anti-tumor macrophages polarization. More importantly, our study reveals that OM@MnO2-PEG-mediated RNT triggers much stronger cGAS-STING pathway-involved immunotherapy than that of EBRT, owing to the duration difference of RT. Therefore, this study develops a powerful sensitizer of radio-immunotherapy and uncovers some differences between EBRT and RNT in the activation of cGAS-STING pathway-related anti-tumor immunity. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-024-02565-7.


Introduction
Radiotherapy (RT) is a fascinating treatment for most cancer and can be further divided into external beam radiation therapy (EBRT) and radionuclide therapy (RNT) [1,2].Notably, RT does not only directly kill the tumor cells by generating various reactive oxygen species, but also triggers the systemic anti-tumor immunity [3,4].The infiltration, polarization and efficiency of various immune cells, including innate and adaptive immune cells, have significantly changed after RT treatment [5][6][7].RT promotes the immunogenic cell death (ICD) and the release of tumor-specific antigen in tumor tissue, together activating DC cells and CD8 + T cells (CTLs) for anti-tumor immunotherapy [8][9][10].More importantly, RT-damaged DNA fragments would leak into the cytoplasm from nucleus or mitochondria, further stimulating cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway [11,12].The cGAS-STING activation shows powerful and positive effect on strengthening the T cells-mediated anti-tumor immunity and reversing M2 macrophage-mediated tumor immunosuppressive microenvironment by secreting type I interferon and various inflammatory cytokines [13][14][15].Meanwhile, as the other side of a double-edged sword, RT up-regulates the expression of various immune checkpoint-related proteins, such as PD-L1, CTLA-4 and IDO-1, and decreases the number of lymphocytes in the body by causing severe bone marrow damage, negatively modulating the anti-tumor immunity [16][17][18][19].Nowadays, both of EBRT and RNT, which possess the same way to trigger biological effects, are needed optimized and considered as important treatment to control the local tumor tissue and stimulate the systemic immune response [20][21][22].The respective source of ionizing radiation in EBRT and RNT leads to the intermittent/shortterm exposure to radiation for EBRT and continuous/ long-term exposure to radiation for RNT.Whether this temporal difference can result in differences in immune stimulation between EBRT and RNT has not been adequately studied.

Graphical Abstract
generation of oxygen affects the energy metabolism of immune cells in the tumor and polarizes the tumor-associated macrophage into the anti-tumor M1 type instead of the pro-tumor M2 type [33][34][35].Besides, related biomaterials have been selected or designed according to the special immune microenvironment induced by RT to strengthen the each process of the T cells-mediated anti-tumor immunity, including antigen release, antigen presentation, DC maturation, T cell infiltration and CTL effect, for systemic anti-tumor adaptive immunotherapy [19,[36][37][38].Notably, bacteria and bacterial outer membrane vesicles (OMVs) can specifically cluster in the tumor area and involve in multiple steps to promote the anti-tumor immunity, mediating perfect combination treatment effect of immunotherapy and RT [39][40][41][42][43]. Additionally, metal ions, including Mn 2+ , Zn 2+ and Hf 4+ , have been reported to enhance the sensitivity of cGAS to cytoplasmic DNA and trigger the production of the second messenger cGAMP, thus playing a key role in the effective and stable activation of cGAS-STING pathway [12,[44][45][46].The existence of these metal ions in the tumor maintains the RT-induced cGAS-STING activation and enlarges the RT-triggered anti-tumor immunity.
In this study, OMVs were collected and biomineralized by manganese oxide (MnO 2 ), obtaining OM@MnO 2 -PEG nanoparticles for enhanced radio-immunotherapy of tumor via increasing the RT-caused ICD and cGAS-STING activation.In the acidic tumor microenvironment, OM@MnO 2 -PEG nanoparticles could react with hydrogen peroxide (H 2 O 2 ) and then produced a plenty of Mn 2+ , O 2 and OM fragments.The relieved tumor hypoxia improved the radio-sensitivity of tumor, amplifying RT-caused ICD and cGAS-STING activation.Meanwhile, oxygen/OMs promoted the macrophage polarization to anti-tumor M1 type and OM fragments enhanced the efficiency of tumor antigen presentation via the recognition of lipopolysaccharide (LPS) and toll-like receptors (TLRs).Notably, the prolonged high content of manganese ions in the tumor maintained the activated condition of cGAS-STING pathway.These comprehensive factors made OM@MnO 2 -PEG significantly enhance the immune stimulation effect of radiotherapy with high bio-safety.Especially, our yielding nanoparticles could improve the immune stimulation of both of EBRT and RNT, and the enhancement degree showed some difference.When the same local treatment outcome was obtained by EBRT and RNT, the distant anti-tumor effect of RNT was better than that of EBRT, which was related to the cGAS-STING pathway activation.In detail, cGAS-STING pathway activation was high and then gradually decreased after EBRT, but remained high in 7 days after 131 I-mediated-RNT Fig. 1a.Therefore, this work developed a novel biomaterial for improving the immune stimulation effect of EBRT and RNT, and discovered some differences in cGAS-STING pathway activation between EBRT and RNT, providing some reference and strategic design for the implementation of clinical tumor RT.

Results and discussion
The preparation and characterization of OM@MnO 2 -PEG OMVs were extracted from attenuated salmonella typhimurium VNP20009 through ultracentrifugation and then modified with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-n-(polyethylene glycol)-5000 (DSPE-PEG 5000 ), yielding OM-PEG nanoparticles.Then, with reference to the reported literatures, [47][48][49] manganese ions were loaded onto the OMs via a biomineralization process to obtain OM@MnO 2 -PEG.Additionally, this nanoparticle could be labeled by radio-iodine Fig. 1b.The collected OMVs possessed lipid-bilayered vesicular structures with the size of 20-200 nm observed by transmission electron microscope (TEM) Figure S1a.In contrast, OM@MnO 2 -PEG showed metallic inorganic matter on the broken OMVs Fig. 1c.The destruction of the complete vesicle structure might be attributed to the hypotonic and ultra-sonication treatments during the preparation of OM@MnO 2 -PEG.Furthermore, elemental mapping of OM@MnO 2 -PEG was carried out and the overlapping of manganese and oxygen indicated the successful formation of MnO 2 on OMs Fig. 1d.Meanwhile, UV-Vis-NIR absorption spectrum of OM@MnO 2 -PEG showed the characteristic absorption peaks of OM-PEG and MnO 2 , doubly confirming the successful biomineralization of MnO 2 onto OMs Figure S1b.The dynamic light scattering (DLS) analyses discovered that the hydrated particle size peak of OMVs and OM@MnO 2 -PEG was about 25 nm and 60 nm, respectively Figure S1c.The SDS-PAGE of OM-PEG and OM@MnO 2 -PEG showed the almost identical protein bands, illustrating the presence of OM protein in OM@MnO 2 -PEG Figure S2.
Finally, in order to test the radioactive iodine labeling stability of OM@MnO 2 -PEG, 131 I-labeled this nanoparticle was incubated in serum and the deciduous 131 I was further measured by a γ-counter.Most of the labeled iodine stayed stable on the nanoparticles after repeated cleaning and centrifugation, indicating our obtaining nanoparticle could be used as an effective carrier of radionuclide iodine Fig. 1e.Besides, the release of Mn 2+ and production of O 2 when OM@MnO 2 -PEG was incubated at different pH and H 2 O 2 content were checked.It has been reported that MnO 2 could react with H 2 O 2 in the acidic condition, leading to the consumption of H 2 O 2 and generation of Mn 2+ /O 2 [47][48][49].As shown in d Elemental mapping analysis of OM@MnO 2 -PEG (scale bar 50 nm).e Radio-labeling stability of radioactive iodine for OM-PEG and OM@MnO 2 -PEG in mouse serum (n = 3).f Release profiles of Mn 2+ from OM@MnO 2 -PEG in buffer solution at different pH (n = 3).g The O 2 concentration changes in acid solution (pH 6.5) with different concentrations of H 2 O 2 .Data are presented as Mean ± SEM Fig. 1f, g, Mn 2+ could be quickly released and significant amount of O 2 was immediately produced at low pH/ high H 2 O 2 conditions, suggesting that OM@MnO 2 -PEG might be given off OM fragments, Mn 2+ and O 2 in tumor microenvironment.

The radio-immunotherapy sensitization effect of OM@ MnO 2 -PEG in vitro
Furthermore, the anti-tumor activity of OM@MnO 2 -PEG was tested in vitro.Firstly, the cell uptake of OM@ MnO 2 -PEG by tumor cells and macrophage cells was measured.OM-PEG could be extensively taken in by both of 4T1 cells and RAW 264.7 cells in a time-dependent manner and the MnO 2 modification slightly reduced the cell uptake of the nanoparticle, reflecting that OM@ MnO 2 -PEG could be used as an effective drug delivery system Fig. 2a, b.The potential cytotoxicity of OM-PEG and OM@MnO 2 -PEG was studied by CCK-8 assay.Both of them showed no notable cytotoxicity to 4T1 cells and RAW 264.7 cells at our test concentrations, indicating the great biocompatibility of OM@MnO 2 -PEG Fig. 2c,  d.Considering the oxygen generation from the reaction between OM@MnO 2 -PEG and H 2 O 2 , OM@MnO 2 -PEG nanoparticle was used to enhance the radio-sensitivity of 4T1 cells.The immunofluorescence intensity of γ-H2AX foci, a biomarker of DNA double-strand breaks, was used to study the extent of DNA damage induced by X-ray treatment.Notably, the immunofluorescence intensity in the 4T1 cells treated by OM@MnO 2 -PEG plus RT was strongest, proving that OM@MnO 2 -PEG was efficient radio-sensitizer Fig. 2e and i, Figure S3a.Moreover, owing to the existence of OM and oxygen, RT-induced ICD might be improved.The expression of calreticulin (CRT) and the release of high mobility group protein B1 (HMGB1) were recognized as the markers of ICD from dying cells.As shown by confocal microscopic images, 4T1 cells treated with OM@MnO 2 -PEG and RT showed significant expression of CRT on the surface of 4T1 cells and obvious release of HMGB1 from the nucleus Fig. 2f, g, j and k, Figure S3b&c.In this way, OM@MnO 2 -PEG enhanced the anti-tumor effect of RT as well as RTinduced ICD.
Additionally, RT can cause the release of damaged DNA fragments from the nucleus/mitochondria into the cytoplasm, further activating cGAS-STING pathway [11,12].Mn 2+ , which can be released from OM@ MnO 2 -PEG in tumor acidic microenvironment, is also proved to enhance cGAS-STING activation [44,47,48].Therefore, we next investigated whether OM@MnO 2 -PEG could enlarge cGAS-STING pathway activation under X-rays exposure in both of 4T1 cells and RAW 264.7 cells.Immunofluorescence was used for validation of the expression of p-STING, which is an activated protein in the cGAS-STING dependent signaling pathway.OM@MnO 2 -PEG plus RT promoted the obvious up-regulation of p-STING expression in both of 4T1 cells and RAW 264.7 cells, demonstrating that OM@ MnO 2 -PEG plus RT could indeed activate cGAS-STING pathway Fig. 2h, l, m and n, Figure S3d, S4.Moreover, the content of interferon-β (IFN-β) as a result of cGAS-STING pathway activation was tested by enzyme linked immune-sorbent assay (ELISA).The results showed that OM@MnO 2 -PEG plus RT could increase the secretion of IFN-β Fig. 2o.Taken together, OM@MnO 2 -PEG could not only enhance the radio-sensitivity of tumor cells, but also strengthen RT-induced ICD and cGAS-STING pathway activation.
The tumor microenvironment regulation by OM@ MnO 2 -PEG Motivated by the perfect sensitizing effect on radioimmunotherapy of OM@MnO 2 -PEG in vitro, we performed the enhanced radio-immunotherapy of tumor in vivo using this nanoparticle.Firstly, in order to understand the in vivo behaviors of OM@MnO 2 -PEG, we quantitatively studied the blood circulation and bio-distribution profiles of 131 I-OM@MnO 2 -PEG. 131I-OM@MnO 2 -PEG was intravenously injected into 4T1 tumors-bearing mice and the blood was collected from these mice at appointed time (0.5, 1, 2, 4, 6, 8, 10 and 24 h) post injection.Then, the radioactivity of these blood samples was determined by a γ-counter.As shown in Fig. 3a, the blood circulation half-life of 131 I-OM@MnO 2 -PEG was long and almost similar with that of 131 I-OM-PEG.For the bio-distribution study, the major organs, including heart, liver, spleen, lung and kidney, and tumors were collected at 24 h post intravenous injection, and their weight and radioactivity were recorded.The tumor accumulation of OM-PEG and OM@MnO 2 -PEG was 7.65% ± 1.60% and 4.94% ± 0.66%, and their liver accumulation was 25.22% ± 1.83% and 12.99% ± 1.12%, suggesting the good tumor-targeting capacity of OM-PEG and OM@MnO 2 -PEG Fig. 3b.However, the amount of Mn 2+ delivered by OM@ MnO 2 -PEG to the tumor was limited and would gradually decrease over time after intravenous injection.This tumor-targeting behavior could image the tumors and outline their location to guide the follow-up local drug administration and RT implement.Optionally, OM@ MnO 2 -PEG could be injected intratumorally with the help of advanced imaging techniques and interventional methods. 131I-OM@MnO 2 -PEG exhibited good tumor-specific retention and low off-target toxicity in one week post intratumoral injection Fig. 3c.Consistent to the result of bio-distribution study, SPECT images showed that 131 I-OM@MnO 2 -PEG had a strong tumor retention ability after intratumoral injection Fig. 3d.Sequentially, inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantitatively examine the bio-distribution of OM@MnO 2 -PEG after being administrated intratumorally by detecting the Mn content in tumors and major organs.A plenty of Mn 2+ was accumulated in the tumor in one week, which was benefit to maintain the activation of cGAS-STING pathway.In addition, a part of Mn 2+ was existed in the kidney, indicating the degradation of OM@MnO 2 -PEG in the acidic tumor microenvironment Fig. 3e.
With the reaction between OM@MnO 2 -PEG and H 2 O 2 , oxygen could be produced in the tumor.Therefore, we checked the tumor hypoxia after different treatments.OM@MnO 2 -PEG could relieve the hypoxic microenvironment of the tumor and the effect of intratumoral injection was more effective than that of intravenous injection.Fig. 3f, Figure S5.

The tumor growth inhibition by OM@MnO 2 -PEG-mediated RT
Next, we explored the tumor radio-sensitization ability of OM@MnO 2 -PEG and the anti-tumor immune-stimulatory effect of OM@MnO 2 -PEG-mediated RT in vivo.Thirty mice bearing subcutaneous bilateral 4T1 tumors were divided into the following six groups at random (n = 5 per group): G1: PBS; G2: OM-PEG; G3: OM@MnO 2 -PEG; G4: X-rays; G5: OM-PEG + X-rays; G6: OM@MnO 2 -PEG + X-rays.The dose of X-rays was 6 Gy and the dose of OMs was 5 μg protein per mouse.The treatment schedule was showed in Fig. 4a.The primary tumors were exposed to the X-rays in half an hour post intratumoral injection of the nanoparticles.Then the tumor size was measured with a vernier caliper and the body weight of mouse was measured every two days.According to the tumor growth curves of primary tumors, OM@MnO 2 -PEG had a certain inhibitory effect on the primary tumor and it could enhance the therapeutic effect of X-rays exposure, demonstrating OM@MnO 2 -PEG was an effective radio-sensitizer Fig. 4b.Moreover, this enhanced RT inhibited the distant tumor growth to some extent, owing to the RT-induced anti-tumor immune-stimulatory effect Fig. 4c.Additionally, there was no remarkable loss in the body weight of mice, suggesting the bio-safety of our therapeutic strategy Fig. 4d.Meanwhile, the hematoxylin-eosin (H&E) stained images showed no obvious toxicity of our treatment strategy to the normal tissues, including heart, liver, spleen, lung and kidney Figure S6.The level of cell damage and apoptosis of the primary/distant tumor sections were also analyzed by H&E staining.As predicted, the treatment of OM@MnO 2 -PEG and X-rays could induce the most serious damage to the tumor cells in both of primary and distant tumor tissue, confirming the excellent radio-sensitization effect of OM@ MnO 2 -PEG Fig. 4e, Figure S7.
RT induced the anti-tumor effect by directly killing the tumor cells and triggering the systemic immune response.Meanwhile, biomaterials could be designed to strengthen both of the cell-killing capacity and the positive anti-tumor immune stimulation of RT.Firstly, RT could cause DNA damage and ICD of primary tumors and OM@MnO 2 -PEG could amplify these effects.We performed the immunofluorescence staining (γ-H2AX, CRT and HMGB1) assay of the primary tumors.OM@ MnO 2 -PEG injection or X-rays exposure could enhance the expression of γ-H2AX/CRT and promote the migration of HMGB1, and OM@MnO 2 -PEG-mediated RT showed the strongest ability to cause these changes Fig. 4f, g and h, Figure S8.Moreover, p-STING expression in the primary tumors was analyzed by immunofluorescence and western blotting.The results showed that OM@MnO 2 -PEG could promote the RT-induced cGAS-STING activation, which could further strengthen RTtriggered anti-tumor systemic immunotherapy of tumor Fig. 4i, j and k, Figure S9.Different with other biomaterials, OM@MnO 2 -PEG combined the respective advantage of OM and MnO 2 in radio-immunotherapy sensitization.This nanoparticle could produce/release OM, Mn 2+ and O 2 in the tumor microenvironment.O 2 improved the treatment outcome of both of radiotherapy and immunotherapy [47][48][49].Mn 2+ can synergy with O 2 -enhanced radiotherapy to activate the cGAS-STING pathway [49].Moreover, OM plays a key role in many aspects of innate and adaptive immunity [40].Therefore, OM@MnO 2 -PEG could enhance the direct toxicity and the efficacy of immune activation of RT.

Phenotype analysis of immune cells
The enhanced ICD and cGAS-STING signaling pathway in the process of RT could promote the formation of an "in-situ" tumor vaccine and reversal of the inhibitory immune microenvironment, further changing the phenotype of various immune cells.To clarify the mechanism, 6 groups of Balb/c mice bearing subcutaneous bilateral 4T1 tumors were prepared and sacrificed 3 days after indicated treatments.The bilateral tumors and adjacent groin inguinal lymph nodes close to the primary tumors were collected for phenotype analysis of immune cells by flow cytometer.The immunoassay of DC maturation was firstly performed.Compared with other treatments, OM@MnO 2 -PEG-mediated RT significantly enhanced the frequency of DC maturation, demonstrating the satisfactory presentation of the tumor antigen in our antitumor strategy Fig. 5a, b Figure S10.Next, we checked the proportion of adaptive immune cells, mainly including CTLs and regulatory T cells (Tregs), and innate immunocytes, including M1-type macrophages and M2-type macrophages, in both of primary and distant tumors.The treatment of OM@MnO 2 -PEG plus X-rays increased the infiltration of CD8 + T cells into primary tumor, while the proportion of immunosuppressive Tregs (CD3 + CD4 + Foxp3 + ) was decreased in CD4 + T cells after this treatment Fig. 5c, d, Figure S11, S12.Additionally, the similar tendency of immune analysis about the CD8 + T cells and Tregs were demonstrated in the distant tumors, suggesting that our treatment strategy could induce powerful and systemic T cells-involved anti-tumor immunity Fig. 5g, h, Figure S13, S14.Moreover, OM@MnO 2 -PEGmediated RT could also activate the innate anti-tumor immunity by promoting the macrophage polarization to M1 in the primary and distant tumors Fig. 5e, f, i and j, Figure S15, S16.This kind of macrophage polarization might be attributed to the integrated function of O 2 , LPS of OMs and cGAS-STING activation in the tumor microenvironment.Taken together, the strong T cells and macrophage-mediated anti-tumor immune response could be realized in mice treated with OM@MnO 2 -PEG-mediated RT.Besides, the content of anti-tumor cytokines, including tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), was also increased after OM@MnO 2 -PEG-mediated RT, indicating the existence of systemic anti-tumor immunity in mice treated with our strategy Fig. 5k, l.In our study, OM@MnO 2 -PEG released OM fragments, Mn 2+ and O 2 in the tumor microenvironment.The relieved tumor hypoxia made tumor cells sensitive to RT, resulting in severe ICD and DNA damage of tumor.DNA fragments in cytoplasm together with detained Mn 2+ caused the activation of cGAS-STING, which played an important role in DC maturation, the proliferation/ tumor invasion of CTLs/Tregs and macrophage polarization.Meanwhile, the production of O 2 and OM fragments in the tumor tissue could also make contribution to enhance the proportion of anti-tumor M1.In this way, OM@MnO 2 -PEG-mediated RT realized multi-stage and multi-angle activation of adaptive immunity and innate immunity.

Comparison of the tumor growth inhibition between OM@ MnO 2 -PEG-mediated EBRT and RNT
RT is divided into EBRT and RNT, according to the different radiation sources.EBRT and RNT shows similar biological mode of action and pathway of sensitization.The irradiation exposure in EBRT is transient and intense, but irradiation exposure in RNT is constant and gentle.Whether the immune stimulation of these two kind of RT has some different characteristics is not clear.Therefore, we preformed 131 I-OM@MnO 2 -PEGmediated RNT and compared the anti-tumor effects between OM@MnO 2 -PEG-mediated EBRT and RNT.We labelled 131 I onto OM@MnO 2 -PEG nanoparticles at different radioactive dose.Mice were divided into five groups: GI: PBS; GII: RNT (20 μCi); GIII: RNT (40 μCi); GIV: RNT (80 μCi); GV: EBRT (6 Gy).The mice were treated as shown in the Fig. 6a, and 131 I-OM@MnO 2 -PEG or OM@MnO 2 -PEG was injected intratumorally.With the increase of the dose of radionuclide, the therapeutic effect of RNT on primary and distant tumors was gradually enhanced, showing the powerful tumor-killing and anti-tumor immunity-stimulating capacity of RNT.Moreover, RNT (40 μCi) exhibited equivalent inhibitory effect on the primary tumors to EBRT (6 Gy), but showed better inhibitory effect than EBRT (6 Gy) on the distant tumors, suggesting that RNT had better immune stimulation effect than EBRT Fig. 6b, c.In addition, the treatment of RNT and EBRT prolonged the survival period of tumor-bearing mice Fig. 6d.The body weight of mice was not remarkably decreased after various treatments, suggesting the perfect bio-safety of these therapeutic strategies Fig. 6e.EBRT took potent effect in a short period of time, while RNT gradually took effect.The time course of RT-induced DNA damage might affect the degree and duration of cGAS-STING activation, further regulating the anti-tumor immunity.Therefore, we compared the degree of the cGAS-STING activation in one week after treatments EBRT and RNT.In detail, we investigated the expression of p-IRF-3 in tumors, cGAMP in spleens and IFN-β in the serum at day 1, 3, 5 and 7 after EBRT or RNT measured by flow cytometer or ELISA.The p-IRF3/ cGAMP was highly expressed at day 1, and then got gradually low at day 3, 5 and 7 in the mice treated with EBRT.In contrast, the expression of p-IRF3/cGAMP kept relatively high and stable in one week in the mice treated with RNT Fig. 6f, g.Similarly, the content of IFN-β in the serum presented the same tendency in mice treated with EBRT or RNT Fig. 6h.Taken together, both of RNT and EBRT could enhance the activation of cGAS-STING and RNT showed a relative longer-term effect on cGAS-STING pathway activation than that of EBRT.
To further validate the participation of cGAS-STING activation in the therapeutic effect of EBRT and RNT, T cells in primary tumors at day 3 post injection (n = 3).e, f Proportion of M1 macrophages (CD11b + F4/80 + CD80 + ) and M2 macrophages (CD11b + F4/80 + CD206 + ) in primary tumors at day 3 post injection (n = 3).g Percentage of tumor-infiltrating CD8 + T cells among CD3 + cells in distant tumors with different treatments at day 3 post injection (n = 3).h Proportion of Treg cells gated on CD3 + CD4 + T cells in distant tumors at day 3 post injection (n = 3).i, j Proportion of M1 macrophages (CD11b + F4/80 + CD80 + ) and M2 macrophages (CD11b + F4/80 + CD206 + ) in distant tumors at day 3 post injection (n = 3).k, l The concentration of TNF-α and IFN-γ in serum were analyzed at day 3 after indicated treatments (n = 3).Data are presented as Mean ± SEM.Statistical significance was analyzed by one-way analysis of variance (ANOVA) with the least significant difference post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) STING inhibitor (C-176) was selected to block the function of cGAS-STING pathway Fig. 6i.Mice were divide into six groups: G1′: PBS; G2′: C-176; G3′: EBRT (6 Gy); G4′: RNT (40 μCi); G5′: EBRT (6 Gy) + C-176; G6′: RNT (40 μCi) + C-176.C-176 significantly promoted the tumor growth of primary and distant tumors in mice treated with EBRT or RNT, indicating the important role of cGAS-STING activation in anti-tumor therapeutic effect of RT Fig. 6j, k.More importantly, the growth of primary tumors in mice treated with C-176 plus RNT was faster than that in mice treated with C-176 plus EBRT, suggesting the therapeutic effect of RNT was more dependent on cGAS-STING activation than that of EBRT.The survivorship curves also showed that C-176 injection decreased the anti-tumor ability of EBRT and RNT Fig. 6l.Finally, the change of average body weight in each group confirmed the safety of OM@MnO 2 -PEGmediated ENRT and RNT Fig. 6m.The cGAS-STING activation-involved tumor immune microenvironment was closely related to the efficacy of immunotherapy.Besides, the difference in anti-tumor immune stimulation triggered by EBRT and RNT might be also affected by other factors, such as ICD enhancement, toxicity of radiation to immune cells and the radiation-induced other changes in tumor microenvironment [3,4,8,9,16].Notably, the effect of various biomaterials on the sensitizing ability of radio-immunotherapy during EBRT and RNT may be different.In this study, the continuous production of oxygen from OM@MnO 2 -PEG could enhance the induction of ICD and DNA damage over a long time during RNT, while over a short time during EBRT.Meanwhile, the direct killing of tumor infiltrating immune cells was persistent during RNT and temporary during EBRT.These differences might further cause the different therapeutic effect of radio-immunotherapy, which need more studies to elucidate.

Conclusion
In summary, we successfully extracted OMVs through ultracentrifugation and loaded MnO 2 onto OMs through biomineralization, yielding OM@MnO 2 -PEG nanoparticles.In the tumor microenvironment with low pH and high H 2 O 2 content, this nanoparticle could release OM fragments, O 2 and Mn 2+ .The generated O 2 relieved the hypoxic tumor microenvironment and alleviated the radiation resistance of tumor tissue.Under X-rays exposure, ICD of the tumor cells would be caused, and a plenty of DNA was damaged and leaked into the cytoplasm, further activating the cGAS-STING pathway.This RT-induced ICD could be amplified by O 2, and OM fragments enhanced the antigen presenting ability of DCs.More importantly, the released Mn 2+ could directly bind to cGAS to enhance the binding affinity between cGAS and cytoplasmic DNA, thereby making cells more sensitive to activate the downstream STING pathway and finally producing strong anti-tumor immunity even in the presence of trace amounts of cytoplasmic DNA.Besides, all of O 2 , OMs and cGAS-STING activation could promote the polarization of macrophages towards anti-tumor M1.Notably, compared to EBRT, RNT could generate more persistent and powerful cGAS-STING activation, and then triggered much stronger systemic anti-tumor immunity.Therefore, a novel nanoparticle was obtained in this study to enhance the direct killing ability and the immune stimulation effect of RT, and this nanoparticle-mediated EBRT and RNT showed differential reinforcement of cGAS-STING pathway-involved immunotherapy.

Fig. 1
Fig. 1 The preparation and characterization of OM@MnO 2 -PEG.a The schematic diagram of the enhanced systemic anti-tumor immunity induced by OM@MnO 2 -PEG-mediated RT. b Schematic diagram showing the synthesis of 131 I-OM@MnO 2 -PEG.c TEM of OM@MnO 2 -PEG (scale bar 100 nm).d Elemental mapping analysis of OM@MnO 2 -PEG (scale bar 50 nm).e Radio-labeling stability of radioactive iodine for OM-PEG and OM@MnO 2 -PEG in mouse serum (n = 3).f Release profiles of Mn 2+ from OM@MnO 2 -PEG in buffer solution at different pH (n = 3).g The O 2 concentration changes in acid solution (pH 6.5) with different concentrations of H 2 O 2 .Data are presented as Mean ± SEM

Fig. 4 Fig. 4 (
Fig.4 The tumor growth inhibition by OM@MnO 2 -PEG-mediated RT. a Schematic diagram of the experimental schedule.The dose of X-rays was 6 Gy.b, c Primary and distant tumor growth curves of mice with indicated treatments (n = 5).d Average body weights of 4T1 tumors-bearing mice after various treatments (n = 5).e H&E staining of primary tumor sections with various treatments (scale bar: 20 μm).Fluorescent images of γ-H2AX (f), CRT (g), HMGB1 (h) and p-STING (i) expression in primary tumor sections with the indicated treatments (scale bar: 25 μm).j, k Representative western blotting images and corresponding statistical analysis of the expression of p-STING/STING in primary tumor with the indicated treatments (n = 3).Data are presented as Mean ± SEM.Statistical significance was analyzed by one-way analysis of variance (ANOVA) with the least significant difference post hoc test.(*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) (See figure on next page.)

(Fig. 5
See figure on next page.)Phenotype analysis of immune cells.a, b Representative flow cytometry plots and quantification of DC maturation in lymph nodes of 4T1 tumor-bearing mice with various treatments (gated on CD11c + DC cells) at day 3 post injection (n = 3).c Percentage of tumor-infiltrating CD8 + T cells among CD3 + cells in primary tumors with different treatments at day 3 post injection (n = 3).d Proportion of Treg cells gated on CD3 + CD4 +

Fig. 6
Fig. 6 Comparison of the tumor growth inhibition between OM@MnO 2 -PEG-mediated EBRT and RNT. a Schematic diagram of the experimental schedule of EBRT and RNT.b, c Primary and distant tumor growth curves of 4T1 tumor-bearing mice after various treatments (n = 5).d The survivorship curves of 4T1 tumor-bearing mice after various treatments (n = 5).e Average body weights of 4T1 tumors-bearing mice after various treatments (n = 5).MST indicated median survival time.f The expression of p-IRF in primary tumor tissues with indicated treatments measured by flow cytometer (n = 3).g The expression of cGAMP in spleens with indicated treatments measured by ELISA (n = 3).h The expression of IFN-β in serum with indicated treatments measured by ELISA (n = 3).i Schematic diagram of the experimental schedule of C-176 inhibition.j, k Primary and distant tumor growth curves of 4T1 tumor-bearing mice after various treatments (n = 5).l The survivorship curves of 4T1 tumor-bearing mice after various treatments (n = 5).MST indicated median survival time.m Average body weights of 4T1 tumors-bearing mice after various treatments (n = 5).Data are presented as Mean ± SEM.Statistical significance was analyzed by one-way analysis of variance (ANOVA) with the least significant difference post hoc test.Statistical significance of survivorship curves was analyzed by log-rank test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) (See figure on next page.)